ABSTRACT
Cyclic GMP-AMP synthase (cGAS) is an endogenous DNA sensor that synthesizes cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP) from ATP and GTP. 2'3'-cGAMP activates the stimulator of interferon genes (STING) pathway, resulting in the production of interferons and pro-inflammatory cytokines. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the phosphodiesterase that negatively regulates the STING pathway by hydrolyzing 2'3'-cGAMP. It has been established that the cGAS-STING pathway plays a major role in inhibiting tumor growth by upregulating T cell response. Herein, we demonstrate that AVA-NP-695, a selective and highly potent ENPP1 inhibitor, apart from the immunomodulatory effect also modulates cancer metastasis by negatively regulating epithelial-mesenchymal transition (EMT). We established that the combined addition of 2'3'-cGAMP and AVA-NP-695 significantly abrogated the transforming growth factor beta (TGF-êµ)-induced EMT in MDA-MB-231 cells. Finally, results from the in vivo study showed superior tumor growth inhibition and impact on tumor metastasis of AVA-NP-695 compared to Olaparib and PD-1 in a syngeneic 4T1 breast cancer mouse model. The translation of efficacy from in vitro to in vivo 4T1 tumor model provides a strong rationale for the therapeutic potential of AVA-NP-695 against triple-negative breast cancer (TNBC) as an immunomodulatory and anti-metastatic agent.
Subject(s)
Programmed Cell Death 1 Receptor , Triple Negative Breast Neoplasms , Adenosine Triphosphate/metabolism , Animals , DNA , Guanosine Triphosphate , Humans , Interferons , Membrane Proteins/metabolism , Mice , Nucleotidyltransferases/metabolism , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/metabolism , Transforming Growth Factor betaABSTRACT
Ecto-nucleotide pyrophosphatase/phosphodiesterases 1 (ENPP1 or NPP1), is an attractive therapeutic target for various diseases, primarily cancer and mineralization disorders. The ecto-enzyme is located on the cell surface and has been implicated in the control of extracellular levels of nucleotide, nucleoside and (di) phosphate. Recently, it has emerged as a critical phosphodiesterase that hydrolyzes cyclic 2'3'- cGAMP, the endogenous ligand for STING (STimulator of INterferon Genes). STING plays an important role in innate immunity by activating type I interferon in response to cytosolic 2'3'-cGAMP. ENPP1 negatively regulates the STING pathway and hence its inhibition makes it an attractive therapeutic target for cancer immunotherapy. Herein, we describe the design, optimization and biological evaluation studies of a series of novel non-nucleotidic thioguanine based small molecule inhibitors of ENPP1. The lead compound 43 has shown good in vitro potency, stability in SGF/SIF/PBS, selectivity, ADME properties and pharmacokinetic profile and finally potent anti-tumor response in vivo. These compounds are a good starting point for the development of potentially effective cancer immunotherapy agents.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Immunotherapy , Lung Neoplasms/therapy , Pyrophosphatases/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Thioguanine/pharmacology , A549 Cells , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Dose-Response Relationship, Drug , Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Female , Humans , Lung Neoplasms/metabolism , Mice , Mice, Inbred BALB C , Molecular Structure , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/therapy , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/metabolism , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Thioguanine/chemical synthesis , Thioguanine/chemistryABSTRACT
Cathepsin D, an aspartyl protease, is an attractive therapeutic target for various diseases, primarily cancer and osteoarthritis. However, despite several small molecule cathepsin D inhibitors being developed, that are highly potent, most of them show poor microsomal stability, which in turn limits their clinical translation. Herein, we describe the design, optimization and evaluation of a series of novel non-peptidic acylguanidine based small molecule inhibitors of cathepsin D. Optimization of our hit compound 1a (IC50 = 29 nM) led to the highly potent mono sulphonamide analogue 4b (IC50 = 4 nM), however with poor microsomal stability (HLM: 177 and MLM: 177 µl/min/mg). To further improve the microsomal stability while retaining the potency, we carried out an extensive structure-activity relationship screen which led to the identification of our optimised lead 24e (IC50 = 45 nM), with an improved microsomal stability (HLM: 59.1 and MLM: 86.8 µl/min/mg). Our efforts reveal that 24e could be a good starting point or potential candidate for further preclinical studies against diseases where Cathepsin D plays an important role.
